Project Summary/Abstract Many organisms exploit the base-pairing potential of RNA and DNA to enable sequence-based resistance mechanisms against viruses and mobile genetic elements. The best known of these mechanisms, RNA interference (RNAi), uses double-stranded RNA to trigger the silencing of specific genes. However, this mechanism has only been documented in eukaryotes. More recently, clustered regularly interspaced, short, palindromic repeat (CRISPR) loci, present in the genomes of many eubacteria and nearly all archaea, have been shown to confer adaptive, heritable, sequence-based immunity against phages. The repeats and spacers present in CRISPR loci encode CRISPR RNAs (crRNAs) that are processed from longer precursor transcripts and serve as guides for this interference pathway. CRISPR loci are accompanied by a set of cas (CRISPR-associated) genes that encode protein components of the underlying enzymatic machinery. However, the molecular mechanisms of crRNA-directed interference are almost completely uncharacterized. We aim to uncover the mechanistic basis for CRISPR interference. We are using the gram-positive pathogen Staphylococcus epidermidis as a model system because of its clinical importance and experimental tractability. Already our work has yielded three major advances: (i) CRISPR loci can function to limit the spread of conjugative plasmids that confer antibiotic resistance in S. epidermidis and Staphylococcus aureus; (ii) the CRISPR pathway in S. epidermidis directly targets incoming DNA and is therefore fundamentally distinct from RNAi; and (iii) crRNAs distinguish untargeted self DNA (the CRISPR locus) from targeted non-self DNA (plasmids and phage genomes) by differential base pairing outside of the spacer region. Our work has advanced our understanding of CRISPR interference, suggested routes towards limiting the spread of antibiotic resistance, validated our selection of S. epidermidis as a model system, and resulted in many strains, plasmids, and assays that are ideal for in-depth analyses of this novel and fascinating pathway. We anticipate that our prospects for exploiting the CRISPR pathway in practical and applied realms will advance in parallel with our understanding of the underlying mechanisms. Accordingly, our proposed studies are designed to uncover new and fundamental aspects of CRISPR interference in S. epidermidis. Importantly, we will combine in vivo and in vitro approaches and capitalize on the synergies between them. In particular, we will (i) define the functional anatomy of the repeat/spacer region and the crRNAs that they encode; (ii) identify and characterize other loci (including any that lie outside of the cas locus) that are required for interference; and (iii) characterize crRNA-containing ribonucleoproteins (crRNPs) and define their properties, components, activities, and precursor-product relationships. This work will clarify the molecular basis of CRISPR interference and illuminate routes toward tapping its potential in the critical battle against antibiotic resistance and bacterial infection.